ABC transporters govern distribution of drugs, lipids, and peptides throughout the human body and play a critical role in multidrug resistance. This proposal aims to understand the entire transport cycle of an ABC transporter and identify rules governing how different ABC transporters select substrates by using a powerful combination approach combining x-ray crystallography and cryo electron microscopy. The approach outlined here examines the mechanistic cycle of an ABC transporter, from substrate binding to conformational changes using a cryo electron microscopy (cryo-EM) approach that has yielded an unprecedented subnanometer resolution structure of an ABC transporter in a collaboration Dr. Tomasiak was part of with Dr. Yifan Cheng. The ultimate goal is a comprehensive model of several human transporters to reveal a quantitative model of human pharmacokinetics and pharmacogenomics that will give a clearer understanding of how these biologically critical molecules are distributed. The candidate, Thomas Tomasiak, has an interest to use the K99 to launch an independent career weaving several biological disciplines and approaches to studying distribution of drugs, lipids, and other hydrophobic compounds. Dr. Tomasiak hopes to become an innovate assistant professor at a leading institution, contributing to our knowledge of drug disposition and multidrug resistance in microbial pathogenesis and cancer. This proposal is centered on a training program in cryo-EM that will help Dr. Tomasiak become an expert in this field and combine it with his background in x-ray crystallography. The combination of membrane protein crystallography and cryo-EM will make a powerful hybrid approach to answer biological questions concerning conformational state changes and complexes in membrane proteins difficult for either technique alone. Part of the proposed research is in development of new tools for the validation of structural models from cryo-EM. The proposal is designed to supplement Thomas's training by integrating his current knowledge of membrane proteins and x-ray crystallography with the burgeoning field of cryo-electron microscopy. Dr. Tomasiak's mentor, Dr. Stroud, and co-mentor, Dr. Cheng are internationally recognized in their fields and Dr. Stroud has supported numerous postdoctoral researchers into the transition to independence. In addition to the co-mentors, Dr. Tomasiak has assembled an advisory committee of senior scientists who will be able to help guide his research and are ideally situated to guide his career progression to an independent position.